The present invention relates in general to the pseudotyping of retroviruses with lymphocytic choriomeningitis virus. In particular, the invention relates to pseudotyping in MLV packaging cells which are optionally env-deleted, or in packaging cells derived from lentiviruses. Preferably, pseudotyping is carried out by infection with LCMV or a preferably env-deleted mutant, or by transfection with an expression plasmid containing the gp-gene of LCMV or a part thereof and optionally, in addition, the np-, the 1- and/or the z-gene of LCMV. The invention also relates to the use of such pseudotypes for the infection of cells, particularly the use in gene therapy.
Retroviral vectors are increasingly being used in the state of the art, for example, for gene transfer in genetic engineering and medical research or in gene therapy approaches (cf. e.g. C. Baum et al. in Seminars in Oncology: Gene Therapy of Cancer: Translational approaches from preclinical studies to clinical implementations., eds. Gerson and Lattime, Academic Press, 1998). The retroviral vectors are mostly derived from murine leukaemia viruses (MLV) and contain all the sequences of the LTR regions required for integration and the "psgr"-element responsible for packaging. The regions coding for the virus proteins are replaced by foreign genes and the control sequences thereof which it would be desirable to introduce into human cells. The vectors are expressed in so-called helper cell lines (packaging cell lines) which contain a copy of a complete retrovirus genome. It synthesises all the proteins required for replication and infection, but is unable to package its genomic virus-RNA into particles because it has a defect in the "psgr"-sequences. If the retroviral vectors are inserted into these helper cells and transcribed, the transgenic mRNA formed is able, by means of the "psgr"-region which is characteristic of it, to interact with the structure proteins of the helper virus and be packaged to particles. The recombinant virions, which possess no genetic information at all for virus components, adsorb on cells by way of their surface proteins, the capsids are taken up in the cytoplasm, and the transgenic RNA is converted to double-stranded DNA and integrated into the host cell genome. The advantage of this system is the stable integration of the foreign genes which are passed on to the daughter cells on division. The non-specific integration at arbitrary sites of the cell genome, which is characteristic of retroviruses, is a disadvantage.
Retroviral vectors impart a stable colinear integration (i.e. without recombinations and rearrangement of the coding sequences in the vector genome) and thereby a long-term expression of the transgene. Long-term gene expression has otherwise been possible hitherto only by means of the episomal herpes virus vectors or the adeno-associated virus vectors (AAV vectors). The packaging systems (packaging cell lines) have not yet, however, been optimised for the latter vector systems. Moreover, AAV vectors have a lower packaging capacity (about 5 kb for AAV compared with about 10-12 kb for retroviral vectors).
In addition to the gene to be transferred, the transgene, packaging lines also express the vector genome which contains retroviral cis elements. The genomic vector transcript does not, therefore, code for retroviral proteins but is inserted in the packaging lines with the aid of the gag-, pol- and env-gene products into a virion which is infectious but not capable of replication. This virion may then be used as a retroviral vector for transferring the transgene integrated into the vector genome into the desired target cells without further proliferation of the vector occurring there. In other words, the viral vector is only able to infect the target cells but is unable to proliferate any further therein.
The development of retroviral packaging systems is already well advanced and vector supernatants that are free from viruses capable of replication can be produced in large quantities under GMP conditions (Good Manufacturing Practice; Directive of the Commission for laying down principles and guidelines of good manufacturing practice (GMP) for certain medicaments for use in humans (91/356/EEC) of 13.6.91). Vectors based on murine leukaemia virus (MLV vectors) have already been used repeatedly in clinical trials (P. Chu et al., J. Mol. Med. 76 (1998) 184-192).
Two fundamental types of retroviral packaging systems are known in the prior art (J. M. Wilson, Clin. Exp. Immunol. 107 Suppl. 1 (1997) 31-32; C. Baum et al. 1998), loc cit.).
MLV packaging cell lines contain the retroviral genes gag, pol and env (FIG. 1) and the sequences required for packaging the retroviral RNA are deleted (C. Baum et al., (1998), loc. cit.).
The second type of known packaging systems is derived from the lentiviruses (R. Carroll et al., J. Virol. 68 (1994) 6047-6051; P. Corbeau et al., Proc. Natl. Acad. Sci. USA 93 (1996) 14070-14075; L. Naldini et al., Science 272 (1996) 263-267; C. Parolin et al., J. Virol. 68 (1994) 3888-3895; J. Reiser et al., Proc. Natl. Acad. Sci. USA 93 (1996) 15266-15271; J. H. Richardson et al., J. Gen. Virol. 76 (1995) 691-696; T. Shimada et al., J. Clin. Invest. 88 (1991) 1043-1047). Lentiviruses are complex retroviruses which, in addition to the gag, pol and env gene products also express a series of regulatory genes. Examples of lentiviruses from which packaging systems were derived are the human immunodeficiency virus (HIV), the xe2x80x9csimian immunodeficiency virusxe2x80x9d (SIV) and the xe2x80x9cfeline immunodeficiency virusxe2x80x9d (FIV). The structure of the lentiviral packaging systems is similar, in principle, to that of the MLV vectors.
An advantage of lentiviral vectors is that they are also able to infect resting cells. In the case of MLV vectors, on the other hand, the vector genome can be transported into the cell nucleus only during cell division, i.e. when the nuclear membrane is dissolved. However, in view of the complex structure of the lentiviral genome, packaging systems derived from lentiviruses have disadvantages which are manifested in a comparatively low titre and relatively poor stability. Due to the complex genome structure, cis and trans elements in the genome cannot be separated clearly from one another. In the packaging constructs that express lentiviral gag, pol and env genes there are also to be found, therefore, important cis-regulatory sequences (e.g. parts of the packaging signal) which must also be contained in the vector genome. Due to these homologies, recombinations between vector genome and the packaging constructs may occur and thus the release of retroviruses capable of replication (e.g. an HIV wild virus which would be highly undesirable), so these systems are not comparable with MLV packaging lines.
All the vector systems known hitherto in the prior art also have some crucial shortcomings which prevent successful use in gene therapy: 1. Retroviral vectors are mostly produced only in inadequate titres and cannot be concentrated any further due to the instability of their envelope proteins. 2. Vector particles cannot be purified without loss of infectiousness due to the instability of their envelope proteins. Such purification is essential, however, as the cell culture supernatants from which vectors are harvested are contaminated by cellular constituents. 3. Due to their envelope proteins, retroviral vectors are inactivated by human serum complement. 4. The receptor for the envelope protein of the classic amphotrophic vectors is expressed on virtually all the cell lines considered. However, many primary human cells such as hepatocytes and haematopoietic stem cells which are attractive targets of gene therapy are deficient in functional amphotrophic receptors, as a result of which transduction is rendered difficult or prevented.
The object of the present invention is, therefore, to provide retroviral packaging systems which do not have the disadvantages of the packaging cell lines known in the prior art.
In particular, the object of the present invention is to provide packaging systems which permit a stable retroviral transfer of transgenes into the target cells, i.e. which lead to stable integration of the transgene into the genome of the target or host cells followed by stable expression of this gene.
The object according to the invention is achieved in that retroviruses are pseudotyped with lymphocytic choriomeningitis virus (LCMV).
The present invention relates, therefore, to a recombinant virion which is preferably transfected with one or more foreign genes, which may be obtained by pseudotyping the virus particle with lymphocytic choriomeningitis virus (LCMV).
The tropism and also the stability of a virus is determined primarily by the envelope protein. Murine retroviruses are able to incorporate not only the MLV-env coded glycoproteins but also envelope proteins of other types of virus into their virus coat. As a result, so-called pseudotypes are produced. Retroviral pseudotype vectors are produced by expression of foreign viral envelope proteins in MLV packaging lines. Conventional MLV packaging cell lines contain the retroviral genes gag, pol and env. Sequences which are necessary for the packaging of retroviral genomic RNA were deleted. A vector is introduced into such packaging lines which contains not only the gene which is to be transferred but also the retroviral packaging sequence and other retroviral cis elements (LTR, leader). The retroviral RNA genome is inserted with the aid of the gag, pol and env gene products into a virion which is infectious but not capable of replication. This virion can then be used as a retroviral vector for the transduction of cells. Pseudotype packaging lines also contain the envelope protein gene of a foreign virus. The pseudotype packaging lines according to the invention contain the envelope protein gene of LCMV, and expression of the LCMV glycoproteins takes place.
The present invention provides for the first time vector systems which may be produced in high titres and concentrated. The vector particles according to the invention can also be purified without or without any substantial loss of infectiousness. Surprisingly, it has become apparent within the scope of the present invention that the pseudotyping according to the invention is not cytotoxic for the packaging cells. Stable packaging cell lines (packaging systems) are thus provided for the first time which permit a stable retroviral transfer of transgenes into the target cells, i.e. which lead to a stable integration of the transgene into the genome of the target or host cells followed by stable expression of this gene.
The cell lines according to the invention are also characterised by a broad, trans-species host cell spectrum (cell tropism). A crucial advantage of the present invention is the fact that individual mutations in the envelope protein of LCMV can lead to a modification of the tropism of LCMV. That is, due to individual point mutations in gp, viruses that are more likely to infect nerve cells become viruses that are more likely to infect lymphocytes or those that are more likely to infect monocytes.
Within the scope of the present invention, LCMV is used for pseudotyping. It is possible or it may even be preferable to use other strains of LCMV instead of the LCMV wild-type. Slight variations in the gp nucleic acid sequence or in the amino acid sequence of the expressed envelope protein in various strains of LCMV may thus alter substantially the cell tropism (host cell spectrum) of LCMV (M. Matloubian et al., J. Virol. 67 (1993) 7340-7349; M. N. Teng, J. Virol. 70 (1996) 8438-8443; King et al., J. Virol 64; 1990, 5611-5616). No such tropism variants in the glycoprotein are found for any of the other retroviral vector systems known hitherto, and a more targeted transduction of the desired cell type is made possible for the first time according to the invention. According to a preferred embodiment of the invention, it may therefore be advantageous to provide packaging systems with various glycoprotein variants (GP variants) for different applications.
Within the scope of the present invention, the starting material is the gp genes of the neurotropic LCMV strain Armstrong, L(ARM) (L. Villarete et al., J. Virol. 68 (1994) 7490-7496) (region coding for SEQ ID NO: 4; compare appendix to the sequence protocol, re SEQ ID NO: 3), and of the haematotropic strain WE (V. Romanowski et al., Virus Res. 3, (1985) 101-114) (SEQ ID NO: 1). Also included according to the invention are variants (tropism variants) of these two strains in which individual amino acids are exchanged in the gp gene product, since the tropism of the virus can thereby be altered.
It is rather probable that xe2x80x9ccryptic splice regionsxe2x80x9d are located in the RNA sequence, since LCMV is an RNA virus without a nuclear phase during the propagation cycle. The removal of such regions (correction for aberrant splicing) can be utilised to achieve an improved expression. Such xe2x80x9csplice-correctedxe2x80x9d variants are also, therefore, included according to the invention.
Pseudotyping can be improved by such optimisation of GP expression, whereby it is possible to dispense with an additional support by means of at least one further LCMV protein. Generally speaking, expression vectors which permit a high, stable gene expression in eukaryotic cells are suitable for the expression of LCMV. The choice of expression vector is, however, crucial for the packaging of the retroviral LCMV pseudotypes only insofar as it must guarantee a high and stable level of expression, i.e. a level of expression which is high enough to permit the formation of pseudotypes and which is durable (stable) without switching off of the promoter occurring.
The following two expression cassettes are particularly preferred according to the invention:
(CMV promoter)xe2x80x94(xcex2-globin-intron-2)xe2x80x94(gp)xe2x80x94(SV40 poly A-signal) and
(EF-lalpha promoter)xe2x80x94(gp)xe2x80x94(poly-A signal of the G-CSF gene)
(S. Mizushima, Nucleic Acids Res. 18 (1990) 5322, T. Uetsuki, J. Biol. Chem. 264 (1989) 5791-5798).
The sequences for the constituents of the expression cassettes are shown in the sequence protocol or are generally well known:
cytomegalovirus promoter (CMV promoter):
(M. Boshart et al., Cell 41 (1958) 521-530; F.
Langle-Rouault et al., Virol. 72 (7) 6181-5 (1998))
betaglobin-intron-2:
(Jeffreys, A. J. et al., Cell 12 (1977) 1097-1108)
SV40 poly A signal:
(M. Boshart et al., Cell 41 (1958) 521-530; F.
Langle-Rouault et al., Virol. 72 (7) 6181-5 (1998))
EF-lalpha promoter: SEQ ID NO: 9
(S. Mizushima, Nucleic Acids Res. 18 (1990) 5322, T.
Uetsuki, J. Biol. Chem. 264 (1989) 5791-5798).
G-CSF poly A signal:
(S. Mizushima, Nucleic Acids Res. 18 (1990) 5322, T.
Uetsuki, J. Biol. Chem. 264 (1989) 5791-5798).
gp (LCMV): compare SEQ ID NO: 1, 3, region coding for SEQ ID NO: 4 (see also Appendix to the sequence listing).
Within the scope of the present invention, the above-mentioned expression cassettes are also therefore included, changes in the relevant nucleic acid sequences being possible as long as the functionality of the expression cassettes remains intact, i.e. their use according to the invention permits pseudotyping of the packaging cells and also does not prevent the transfection of the target cells and the stable integration of the transgenes into the host genome.
Moreover, an episomal EBV expression vector (Epstein-Barr-Virus; cf. F. Langle-Rouault et al., Virol. 72 (7) 6181-5 (1998)) (pCep4) from Invitrogen also exhibits high expression and is therefore preferred within the scope of the present invention.
The present invention also provides, therefore, a packaging cell which contains the retroviral genes gag (region coding for SEQ ID NO: 12; cf. Appendix to the sequence listing, re SEQ ID NO: 11), pol (region coding for SEQ ID NO: 13; cf. Appendix to the sequence listing, re SEQ ID NO: 11) and optionally the retroviral gene env (region coding for SEQ ID NO: 14; cf. Appendix to the sequence listing, re SEQ ID NO: 11) and/or regulatory retroviral genes (in the case of lentiviral packaging systems, see below, e.g. the gene coding for the lentiviral Rev protein which prevents splicing of the retroviral genomic RNA) and also contains the gene gp coding for the glycoproteins GP-1 and GP-2 of LCMV (region coding for SEQ ID NO: 4; cf. Appendix to the sequence listing, re SEQ ID NO: 3) or a part thereof. Also included are nucleic acid sequences which exhibit modifications or deviations (mutations, deletions etc.) in the sequences as long as, when used according to the invention, the pseudotyping of the packaging cells is guaranteed and the transfection of the target cells and the stable integration of the transgenes into the host genome is not impeded. This includes fragments of the named sequences. These derivatives should always be included hereinafter when any gene as such is mentioned.
Within the scope of the present invention, xe2x80x9cGPxe2x80x9d or xe2x80x9cGP proteinxe2x80x9d denotes the GP-C precursor protein from which GP-1 and GP-2 are then produced by proteolytic cleavage, these being denoted hereinafter simply xe2x80x9cLCMV glycoproteinxe2x80x9d.
According to the invention, moreover, pseudotype packaging systems are provided in which, apart from the gp gene product (SEQ ID NO: 4), one or more other genes of LCMV are expressed such as, for example, the gene np coding for the nucleoprotein (region coding for SEQ ID NO: 5; cf. Appendix to the sequence listing, re SEQ ID NO: 3), the gene z coding for a protein with an unknown function (region coding for SEQ ID NO: 8; cf. Appendix to the sequence listing, re SEQ ID NO: 6) and the gene 1 coding for RNA polymerase (region coding for SEQ ID NO: 7; cf. Appendix to the sequence listing, re SEQ ID NO: 6). According to a particular embodiment of the invention, these genes may stem either from the WE or Armstrong strain of LCMV. In this connection, either the complete sequences of the genes np, z and/or 1 (SEQ ID NOs: see above) or parts thereof may be used. Sequences included according to the invention are nucleic acid sequences which exhibit modifications or deviations (mutations, deletions etc.) in the sequences (derivatives), as long as pseudotyping of the packaging cells is guaranteed and the transfection of the target cells and the stable integration of the transgenes into the host genome is not impeded. This includes fragments of the named sequences. These derivatives should always be included hereinafter when any gene as such is mentioned.
The invention also provides, therefore, a packaging cell which, in addition to the gp gene of LCMV, contains at least one gene from the group comprising the gene np coding for the nucleoprotein, the gene 1 coding for RNA polymerase and the gene z of LCMV coding for a protein of unknown function.
The MLV/LCMV pseudotypes, i.e. recombinant retroviral virions which contain the LCMV glycoprotein incorporated in their coat, are produced by the packaging cells according to the invention.
For the production of the recombinant virions, the starting materials for the viral packaging cell lines within the scope of the present invention are preferably all the cell lines that produce high titres of retroviral vectors. Cell lines used in preference are NIH3T3, Te671, 293T, HT1080 (F. L. Cosset et al., J. Virol. 69 (1995) 7430-7436; D. Markowitz et al., Virology 167 (1988) 400-406); W. S. Pear et al., PNAS 90 (1993) 8392-8396). The choice of cell line is not important, however, for the specific advantages of the invention because it has become apparent that GP does not have a toxic effect in any cell line examined hitherto. If, therefore, lines should be found in the future which permit a more efficient vector production (i.e. more stable titre which is at least as high as in the above-mentioned lines,  greater than 106/ml), these may also be used.
The gag and pol genes of the Moloney strain of murine leukaemia viruses (MOMLV) are expressed in the packaging systems used in preference for pseudotyping according to the invention (gag: region coding for SEQ ID NO: 12, pol: SEQ ID NO: 11, Nukleotide 1970-5573; cf. Appendix to the sequence listing, re SEQ ID NO: 11). According to the invention, however, other gag and pol variants of MLV are also included as long as they exhibit the above-mentioned advantages for vector production. In particular, the above-mentioned gene derivatives are included according to the invention.
It was ascertained within the scope of the present invention that LCMV-GP also pseudotypes lentiviral nucleocapsids (see examples). According to a particular embodiment, the packaging systems may also therefore contain the gag and pol gene products of lentiviruses, i.e. lentiviral packaging systems (packaging cell lines) may be used. It is immaterial from which lentivirus the packaging system is derived. Suitable lentiviral packaging cells within the scope of the present invention include, for example, cell lines derived from human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV) or feline immunodeficiency virus (FIV). In this connection, it could be necessary for an efficient production of infectious lentivirus vectors to express, in addition, accessory lentiviral genes such as rev (region coding for SEQ ID NO: 21; cf. Appendix to the sequence listing, re SEQ ID NO: 15) or tat (region coding for SEQ ID NO: 20; cf. Appendix to the sequence listing, re SEQ ID NO: 15) in the case of HIV vectors. Within the scope of the present invention, LCMV proteins may be used for pseudotyping in all the lentiviral packaging systems.
The present invention also provides a (viral) packaging cell or a pseudotyped virion in which the virus is selected from the family of retroviridiae, particularly the MLV-related viruses and the lentiviruses. According to a particular embodiment of the invention, the retroviral packaging cell is selected from the group comprising packaging cells derived from MLV, HIV, SIV and FIV.
According to the invention, moreover, a pseudotyped virion is provided which can be obtained by pseudotyping a retroviral cell of MLV which does not express an ENV protein, the defective mutant L(ARM) of LCMV being used for pseudotyping. Alternatively, other variants of LCMV may also be used.
The present invention also provides a process for the production of the packaging cells/packaging cell lines according to the invention, wherein a retroviral packaging cell line transfected with one or more foreign genes is infected with LCMV according to known methods (cf. e.g. Maniatis, Sambrook, Fritsch: Molecular cloning, a laboratory manual; Cold Spring Harbor Laboratory Press, 1982). The virions which contain LCMV glycoprotein incorporated in their coat are expressed by the packaging lines. According to a particular embodiment of the invention, the pseudotype-producing packaging cells may be produced by transfecting a retroviral packaging cell line transfected with one or more foreign genes with an expression plasmid which contains the gp gene of LCMV or a part thereof and optionally, in addition, one or more genes from the group comprising np, 1 and z of LCMV.
The present invention also relates, therefore, to a process for the production of retroviral pseudotype vectors wherein retroviral packaging cells containing at least the gp gene of LCMV or a part thereof (see above) are produced initially and these are then cultivated under conditions that are suitable for the production of virions.
Within the scope of the present invention, the retroviral packaging cell line used is preferably an MLV packaging cell line which does not express a functional ENV protein and the LCMV used for pseudotyping is the deleted mutant L(ARM). The cell line described by von Laer et al. (J. Virol. 72 (1998) 1424-1430) may also be used as the ENV-negative packaging cell line.
The virions or packaging cells lines according to the invention may be used advantageously for producing viral pseudotype vectors which may be used advantageously for the transduction of cell lines but also of primary eukaryotic cells for research purposes (in vitro) or within the context of gene therapy.
The use of virions/packaging cells for gene therapy also comes into consideration, however, within the scope of the present invention. In this connection, gene therapy may include the treatment of infectious diseases such as AIDS and neoplasms such as breast cancer or melanoma and other diseases accessible by gene therapy. The transduction of haematopoietic stem cells also comes into consideration within the scope of the present invention.
The recombinant virion or the recombinant packaging cell (cell line) of the present invention comprises one or more transgenes. Preferably, said transgenes are selected from the group comprising marker genes such as, e.g. neo, lacZ or enhanced green fluorescent protein (EGFP) and/or genes which may be used therapeutically, such as, e.g. the suicide gene herpes simplex virus thymidine kinase (HSV-tk), cytosine deaminase (CD) and sequences with an antiviral effect such as ribozymes, antisense sequences and transdominant-negative genes and genes that may be used in tumour therapy such as mdr-1 for the protection of haematopoietic cells in chemotherapy, and cytokine genes. Moreover, all transgenes which could be of interest within the context of a targeted gene transfer and the expression of the transgene(s) in cells in vitro or within the context of gene therapy may, however, be used.
The invention also includes a process for the preparation of a pharmaceutical preparation for gene therapy wherein viral pseudotype vectors or retroviral packaging cells according to the invention are formulated optionally with pharmaceutically compatible excipients and/or carriers. The present invention also provides a pharmaceutical preparation for gene therapy which comprises retroviral packaging cells according to the invention and optionally pharmaceutically compatible excipients and/or carriers.
The present invention provides for the first time vector systems which may be produced in high titres and concentrated. The vector particles according to the invention may also be purified without or without any substantial loss of infectiousness. The cell lines according to the invention are also characterised by a broad, trans-species host cell spectrum (cell tropism). Surprisingly, it has become apparent within the scope of the present invention that the pseudotyping according to the invention is not cytotoxic for the packaging cells. Stable packaging cell lines (packaging systems) are thus provided for the first time which permit stable retroviral transfer of transgenes into the target cells, i.e. which lead to a stable integration of the transgene in the genome of the target or host cells followed by stable expression of this gene.